4398
Carbon-13 Nuclear Magnetic Resonance of 5-Substi tuted Uracils’ Paul D. Ellis,” R. Bruce Dunlap,” Andrew L. Pollard, Kurt Seidman, and Alan D. Cardin2 Contributionf r o m the Department of Chemistry, University of South Carolina, Columbia, South Carolina 29208. Received June 16, I972 Abstract: 13Cchemical shifts and coupling constants are reported for 17 5-substituted uracils. Overall, the chemical shifts at C-5 and C-6 of the 5-substituted uracils exhibit no obvious correlation with substituent electro-
negativity. Instead, when the 5-substituted uracils are considered as trisubstituted ethylenes, the chemical shift data are shown to be rationalized in terms of the ability of the C-5 substituent to behave as a mesomeric acceptor or donor. It is also demonstrated that the correlation of the chemical shifts at C-6 can be used to identify two categories of 5-substituted uracils whose parent deoxynucleotide derivatives are inhibitors of the enzyme, thymidylate synthetase. It is suggested that nmr spectroscopy is a potentially useful tool for predicting the effectiveness of certain modified substrates as enzymaticinhibitors.
U
racil and certain of its 5-substituted analogs are involved in a number of biochemically significant roles ( i e . , constituents of RNA and DNA) in all living systems. The 5-substituted uracils and their nucleoside and nucleotide counterparts function either as substrates, products, or inhibitors of certain enzymes which catalyze the synthesis, degradation, or interconversion of pyrimidine compounds.3 Certain naturally occurring uracil derivatives are known to participate in intracellular mechanisms regulating pyrimidine metabolism, while a number of synthetic 5-substituted uracils and their derivatives are of value as chemotherapeutic agents. 8 , 4 The investigation of the 5-substituted uracils reported herein is motivated by our basic interest in the mechanistic aspects of enzymes such as thymidylate synthetase, thymidine kinase, and dihydrouracil dehydrogenase, which interact with the uracils or their nucleoside or nucleotide derivatives. For example, three deoxynucleotide derivatives of the 5-substituted uracils, namely deoxyuridine 5’-monophosphate (dUMP), thymidine 5’-monophosphate, and 5-fluorodeoxyuridine 5 ‘-monophosphate, interact with thymidylate synthetase in the role of substrate, product, and inhibitor, respective1y.j Often important insight relative to the mechanism of action of an enzyme can be gathered by examining the factors which are responsible for the mode and degree to which substrates or their analogs bind to the catalyst. In many cases, the nature and position of substituents on a substrate molecule are correlated with both the extent to which such compounds interact with enzymes and their ability to function as substrates or inhibitors in an enzymatic reaction.‘j Acknowledgment of the factors operative in substrate binding is also of great value in the design of affinity labeling reagents and inhibitors of possible chemotherapeutic utility. (1) Presented in part at the 162nd National Meeting of the American Chemical Society, Washington, D. C., Sept 1971. (2) National Science Foundation Undergraduate Research Participant, 1971. (3) P. Roy-Burman, “Analogues of Nucleic Acid Components,” Springer-Verlag, New York, N. Y., 1970. (4) C. Heidelberger, Progr. Nucl. Acid Res. Mol. Biol., 4,l (1965). ( 5 ) R. L. Blakley, “The Biochemistry of Folic Acid and Related Pteridines,” North-Holland Publishing Co., Amsterdam, 1969. (6) M. Dixon and E. C. Webb, “Enzymes,” 2nd ed, Academic Press, New York, N.Y., 1964.
nmr specIn the present study, we have utilized troscopy to characterize 17 5-substituted uracils with respect to their chemical shifts and JCsHcoupling constants. Tarpley and Goldstein7 have used a similar technique to study uracil and the halouracils (see Discussion), and other groups have applied this method to determine and assign the 13C nmr spectra of the naturally occurring nucleosides and nucleotides.* The variations in chemical shifts at C-5 and C-6 as a function of the substituent show a relatively poor correlation with respect to substituent electronegativity, and instead are rationalized in terms of the ability of the substituent to behave as a mesomeric donor or acceptor. Finally, we speculate on the utility of 13C ninr studies of 5-substituted uracils in predicting the effectiveness of the deoxynucleotide analogs as inhibitors of the thymidylate synthetase reaction.
Experimental Section Materials. Uracil and its 5-nitr0, thio, amino, fluoro, methyl, hydroxymethyl, and hydroxy derivatives were purchased from Sigma Chemical Co. (St. Louis, Mo. 63178). Uracil-5-carboxylic acid was purchased from Aldrich Chemical Co. (Cedarknolls, N. .I. 07927), and 5-trifluoromethyluracilwas a gift from Dr. Daniel V. Santi, Department of Chemistry, University of California, Santa Barbara, Calif. 93106. These compounds did not need further purification and were used as obtained. 5-Formyluracil was prepared by the oxidation of 5-hydroxymethyluracil by potassium persulfate in the presence of a catalytic amount of silver nitrate as described by Brossmer and Zieg1er.O The methyl ester of uracil-5-carboxylic acid was synthesized by refluxing the corresponding acid chloride in absolute methanol.lo 5Cyanouracil was prepared by the sequence of reactions described by Prystas and Sorm,“ and 5-methoxyuracil was prepared by the method outlined by Chesterfield.12 Proton nmr and thin layer chromatography were used to characterize the purity of the compounds employed in this research. Two thin layer systems were used here: (A) a tertiary mixture of t e r f (7) A. R. Tarpley, Jr., and J. H. Goldstein, J . Amer. Chem. Soc., 93,3573 (1971). (8) (a) D.E. Dorman and J. D. Roberts, Proc. Nul. Acud. Sci. U.S., 65, 19 (1970); (b) A. J. Jones, M. W. Winkley, D. M. Grant, and R. K . Robins, ibid., 65, 27 (1970); (c) A.J. Jones, D. M. Grant, M. W. Winkley, and R. K. Robins, J . Amer. Chem. Soc., 92, 4079 (1970); and (d) J . Phys. Chem., 74,2684 (1970). (9) R. Brossmer and D. Ziegler, TefrahedronLetf.,43,5253 (1966). (10) H. Gershon, J. Org. Chem., 27,3507 (1962). (11) M. Prystas and F. Sorm, Collect. Czech. Chem. Commun., 31, 3990 (1966). (12) J. H.Chesterfield, J. F. W. McOmie, and M. S. Tute, J . Chem. Soc., 4590 (1960).
Journal of the American Chemical Society / 95:13 / June 27, 1973
4399
Table I. Carbon-13 Chemical Shifts andJCa for the 5-SubstitutedUracilsm Substituent Fd
OCH a' OH NOn NHz CHzOHf CHm CHS~ SI-2
c1
C02CH3h CFa' COzHi Hd Brd CNk Id
6C2b
6C4b
6C6b
aceb
6c2"
6c4"
6C5C
-109.48 -110.19 -109.20 - 109.88 -109.00 - 110.47 - 109.76 - 110.67 -110.35 -109.57 - 109.88 -109.88 -109.60 -110.95 -109.77 -109.36 -110.25
- 117.81 -120.31 -120.59 -115.71 -20.90 - 122.89 -121.78 -124.08 -121.38 -119.25 - 119.40 - 119.20 -122.49 -123.76 -119.39 -120.39 - 120.84
-99.25 -95.40 -91.04 -85.56 -75.88 -72.00 -69.62 -66.96 -65.93 -65.52 -62.48 -61.36 -69.61 -59.78 -53.98 -46.49 -27.14
-85.64 -82.11 -79.92 - 107.78 -81.12 -97.30 - 108.49 -96.91 -107.10 -99.10 -108.88 -102.94 - 109.40 -101.63 - 101.56 -111.26 -106.42
1.47 0.76 1.75 1.07 1.95 0.48 1.19 0.28 0.60 1.38 1.07 1.07 1.35 0.00 1.18 1.59 0.70
5.95 3.45 3.17 8.05 2.86 0.84 1.98 -0.32 2.38 4.51 4.36 4.56 1.27 0.00 4.37 3.37 2.92
-39.47 -35.62 -31.26 -25.78 -21.34 -12.22 -9.84 -7.18 -6.15 -5.74 -2.70 -1.58 -0.93 0.00 5.80 13.09 32.64
6 C 6'
15.99 19.52 21.71 -6.15 25.75 4.33 -6.86 4.72 -5.47 2.53 -7.25 -1.31 -7,77 0.00 0.07 -9.63 -4.79
JC~E
182.0 180 178 186 178 178 178 177.8 183 184.0 180 182.5 185 180.5 184.7 185 184.5
a All chemical shifts and coupling constants are reported in parts per million and hertz, respectively. Chemical shifts of the uracil carbons with respect to internal DMSO. The lac chemical shift of DMSO is -40.5 ppm with respect to TMS. Chemical shifts of the ith carbon in the 5-substituted uracil with respect to the ith carbon in uracil. The chemical shifts of the H , CHI, F, C1, Br, and 1 compounds have been previously reported by Tarpley and Goldstein.' Their values for the chemical shifts agree with ours for the H, CH,, and F compounds within experimental error. The values reported for C1, Br, and I are theirs after converting to a DMSO chemical shift scale, Likewise, the values of Jcea for these compounds are those of Tarpley and Goldstein. The chemical shift of the methyl carbon is - 17.71 ppm for the methyl is 147 Hz. f The chemical shift of the CH2 carbon is -15.19 ppm with respect to DMSO, and with respect to DMSO, and JCE JCHfor the methylene is 145 Hz. 0 The chemical shift of the COH carbon is - 145.66ppm with respect to DMSO, and JCA is 180Hz. * The chemical shifts of the carbonyl and methyl carbons are - 122.57 and -10.99 ppm, respectively, with respect to DMSO. JCEfor the methyl carbon is 147.5 Hz. The chemical shift of the CFa carbon is -82.31 ppm with respect to DMSO, and JCFis 270 Hz. i The chemical shift of the carbonyl carbon is - 124.47 ppm with respect to DMSO. k The chemical shift of the nitrile carbon is -73.98 ppm with respect to DMSO.
butyl alcohol, methyl ethyl ketone, ammonium hydroxide, and water in the ratios 40: 30: 10 :20, respectively, and (B) a mixture of benzene, pyridine, and acetic acid in the ratios of 16:4:1, respectively. All the compounds used gave a single spot on Brinkmann Instruments (Westbury, N. Y. 11590) 0.1" cellulose MN 300 U V Z 6 4 prepared thin layer plates. The compounds synthesized herein gave the following Rfvalues: 5-formyluracil (A) 0.78, (B) 0.36; 5-cyanouracil (A) 0.66, (B) 0.24; 5-methoxyuracil (A) 0.28, ('B) 0.48; methyl ester of uracil-5-carboxylicacid (A) 0.32, (B) 0.21. "r Measurements. The 1aC nmr spectra were determined on a Varian XL-100-15 nmr spectrometer operating in the Fourier transform mode. All the samples were dissolved in DMSO-d6 with approximately 2% DMSO added to each sample. The concentration of the substituted uracils was 0.1 it4 for chemical shift determinations and 0.2 M or higher for evaluations of J c 6 ~ . The DMSO-& furnished the internal lock while the DMSO was used for a chemical shift reference. The precision in the reported chemical shifts and coupling constants is 0.05 ppm and 1 Hz, respectively. The DMSO-& (99.573 was obtained from Diaprep and it was used without further purification.
Results The 13Cchemical shifts and JcsH coupling constants of 17 5-substituted uracils (see structure I for number-
I ing system) are presented in Table I. The order of presentation in Table I is that of increased shielding of carbon 5. The chemical shifts are reported in two ways: (1) in ppm with respect to internal DMSO, and (2) the chemical shift of carbon i in the 5-substituted compound is reported with respect to the analogous carbon in uracil. The latter method of reporting chemical shifts will prove very useful in subsequent discussions concerning substituents effects.
Discussion Overall Trends. The 13C chemical shifts presented in Table I have the expected order of sensitivity to the presence of a C-5 substituent, namely, C-2 < C-4 < C-6 < C-5. The observed ranges of 13C chemical shifts for the systems studied are 2, 6 , 72, and 34 ppm, for C-2, C-4, C-5, and (2-6, respectively. Examination of the coupled and completely 'H noise decoupled 13C spectra of the various uracils in conjunction with previous work leads to a completely unambiguous assignment of the various 13Cresonances.8 Tarpley and Goldstein' were able to successfully correlate the 13Cchemical shifts in a limited number of 5-substituted uracils with substituent electronegativity. It is of interest to see if this overall correlation holds for the 12 additional substituents reported here. A plot of substituent electronegativity, E,, us. the 13C chemical shifts of carbons 5 and 6 is presented in Figure 1. The E, values are those of Dailey and Schoolery. l 3 It is clear from Figure 1 that the linear relationship between 13C chemical shifts and E, that Tarpley and Goldstein obtained no longer holds for a more complete list of substituents. In fact, there is no obvious relationship between the l3C chemical shifts of C-5 and C-6 in the 5-substituted uracils und the substituent electronegativity parameters of Dailey and Schoolery. In view of these results, we must look elsewhere in an attempt to rationalize the observed patterns of chemical shifts. It is convenient to consider the 5-substituted uracils as either a set of trisubstituted ethylenes or monosubstituted benzenes. This is not an unreasonable premise for discussion since the trends in the 13Cchemical shifts reported here are not unlike those that have (13) B. P. Dailey and J. N. Schoolery, J . Amer. Chem. SOC.,77, 3977 (1955).
Ellis, et al.
Nmr of 5-Substituted Uracils
IaC
4400
I I cs:
H
E2 o n -
Y
0
C6: +
41
0
f
8 lL
v)
I-
20
k
I v)
IO--
0
LL
+F
I
v) ~
0.0-
Q
0
5I 0
, .. -0 -10 I--!
-10
0.0
IO
20
30
I3C CHEMICAL SHIFTS OF C 6 (ppm) OOH
-401
OF
Figure 3. A plot of the 13C chemical shifts of C-6 in 5-substituted uracils us. the chemical shifts of the ortho carbon in an analogous series of monosubstitutedbenzenes.
4 3
2
4
SUBSTITUENT ELECTRONEGATIVITY, E,
Figure 1. A plot of substituent electronegativity, E,, us. the 13C and C-6 (+) in 5-substituted uracils. The chemical shifts of C-5 (0) chemical shifts are with respect to the analogous carbon in uracil.
v u"
I1
20
b rn
I-
0.0
e -20 I V
-40
Here, X, and Xd represent mesomeric acceptor and donor groups, respectively.
-20
0.0
20
40
I3c CHEMICAL SHIFTS OF c 5 (ppm)
Figure 2. A plot of the 13C chemical shifts of C-5 in 5-substituted uracils US. the 13C chemical shifts of the substituted carbon in an analogous series of monosubstitutedbenzenes.
been reported earlier for substituted benzenesl4~15 and vinyl compounds.16-1a The overall trends in the lacchemical shifts of monosubstituted benzenes and simple vinyl compounds can be rationalized in terms of mesomeric arguments that reflect the competition between the carbon bearing the substituent, C,, to form a double bond with the atom (or group) X, or C, and the possible ionic character in these bonds. This argument is represented schematically in I1 and 111. (14) G. B. Savitsky and K. Namikawa, J. Phys. Chern., 67, 2754 (1963). (15) G. E. Maciel, ibid., 69, 1947(1965). (16) G. E. Maciel and J. J. Natterstad, J. Chem. Phys., 42, 2427 (1965). (17) G. B. Savitsky, P. D. Ellis, K. Namikawa, and G. E. Maciel, ibid., 49,2395 (1968).
(18) G. E. Maciel, P. D. Ellis, J. J. Natterstad, and G. B. Savitsky, J. Mugn. Resonunce, 1,589 (1969).
Journal of the American Chemical Society / 95:13
111
It is important to ascertain if there is a close linear relationship between the 13C chemical shifts of C-5 and C-6 in the uracils with those for C, and C, in the monosubstituted benzenes. If such a relationship exists, one could, therefore, imply that whatever mechanisms are operational in determining the 13Cchemical shifts in monosubstituted benzenes are also present to about the same extent in the 5-substituted uracils. Furthermore, if there is a good correlation between the above mentioned chemical shifts, then we can apply the mesomeric arguments depicted in structures I1 and 111 to rationalize the observed trends in the 13C chemical shifts in the 5-substituted uracils. Plots of the 13C chemical shift of C-5 us. C, and C-6 VS. C, are presented in Figures 2 and 3, respectively. Note the excellent linear relationship between the lacchemical shifts of C5 and C,. However, the analogous plot for C-6 and C, has considerably more scatter to it. This increased scatter undoubtedly represents a limited breakdown in the assumption that structures I1 and I11 can completely represent the electronic changes in the substituted uracils or substituted benzenes. The data presented in Figure 3 indicate that carbon-6 in a substituted uracil is significantly different than the ortho carbon in a substituted benzene with regard to the ability of the substituent to alter the electron distribution and hence the 13C chemical shift of this carbon. The methods of Savitsky, Ellis, Namikawa, and Maciell' can be employed to describe the factors which contribute t o the l3C chemical shifts of C-5 and C-6 in the substituted uracils. However, before we examine these chemical shifts in detail, let us consider a brief summary of the model used by Savitsky, et al., to describe the l3C chemical shifts in the vinyl systems.
June 27, 1973
Savitsky and coworkers employed the Karplus and Poplelg formulation of 13Cchemical shifts in an attempt t o rationalize the patterns of 13C chemical shifts in unsymmetrically substituted ethylenes in terms of the mesomeric arguments depicted by structures I1 and 111. Briefly, the Karplus-Pople treatment of chemical shifts can be described by eq 1. In this expression, uA
+ gpAA + B#A
=
=AB
+ ,+,ring
(1)
UP is the local diamagnetic term,
up*A the local paramagnetic term, uABis the contribution from currents on other atoms to the chemical shift of atom A, and &,ring is the contribution to the shielding of A from “ring currents.” By local, we mean atom A’s contribution to its own shielding. It is well known that the local paramagnetic term, upAA,dominates the 13C chemical shifts. 19--21 The local paramagnetic term, upAA,in the Karplus-Pople treatment is given by eq 2 upAA
=
- [ e 2 n 2 / 2 m 2 ~ 2 A E ] ( r -B3 )QAB 2pC
(2)
where
and (r-3)2p = (1/24ao?)(3.25 - 0 . 3 5 q ~ ) ~
(4) In these equations, AE represents a “mean electronic excitation” energy, ( Y - ~ ) , ~is the mean inverse cube of the distance from the nucleus for an electron in a 2p orbital, 4~ is the net charge on carbon A, and the P,, are elements of the first-order density matrix in the neglect of overlap approximation. The KarplusPople treatment of chemical shifts has severe limitations in its applicability.21~22However, for a limited series of analogous compounds where the A E parameter can be considered a constant, useful information can be obtained in terms of variations in the (r-3)zp term and the elements of the density matrix. Using eq 2-4, and a set of ir-molecular orbitals with an explicit dependence in terms of parameters that reflects a sort of competition between C, and atom X (or group) to form ir bonds to carbon C,, while also showing the ionic characters in these bonds, Savitsky and coworkers l 7 derived the following expressions for upAA
+ (*/e)
upAA= - 130[0.677 - 0.323f(I - 1){2
[(I
+ 1)(1
- I)fJ1’z]
X
for donors (5)
(19) M. Karplus and J. A. Pople, J. Chem. Phys., 38,2803 (1963). (20) J. A. Pople, ibid., 37,53 (1962). (21) P. D. Ellis, G. E. Maciel, and J. W. McIver, Jr., J. Amer. Chem. SOC.,94,4069 (1972). (22) P. D. Ellis, Ph.D. Thesis, University of California at Davis, 1970.
upAA= -130[1.323 - 0.323f(I+
[(I
+ 1)(1 - Z).f]1’2}
1)]{2
+ (‘/B)X
4401
for acceptors (6)
Here, the parameter I determines the extent of bond ionicity, and f is a measure of the relative importance of C,-to-C, R bonding in comparison to the C,-to-X 7r bonding. Carbon-6 in Substituted Uracils. Inherent in Figure 3 is the ambiguity of correlating relative substituent effects of 13C chemical shifts in terms of substituent electronegativity alone. The line drawn in Figure 3 goes through the halogens. When X is CH20H, CH3, OMe, and H, the 13C chemical shifts of carbon-6 fall on or very near the line through the halogens. Whereas, when X is OH, NHz, CN, C 0 2 H , and NOz, their 13C chemical shifts depart dramatically from the line. Fluctuations in bonding and charge density within the u framework could be responsible for the observed trends in the 13Cchemical shifts of carbon-6. If this was the case, these changes in bonding should be reflected in the carbon-hydrogen coupling constant, JCH.Examination of Table I yields no obvious relaand the chemical shifts of carbon tionship between JCH 6. However, these departures from the line can be correlated with the ability of the substituent t o be either a mesomeric donor or acceptor. When X is a mesomeric donor group, there are positive deviations from the line and when X is an acceptor group there are negative deviations from the line. When X is F, OMe, OH, and NH2, one could consider the I values to be approximately the same for the latter four substituents. That is, it is assumed that these substituents can polarize the C-X u bond to about the same extent. Within the scope of this model, the deviations from the halogen line can arise because of different f values for the substituents. From eq 5, it can be seen that for a given I value, I 2 1, as f decreases the paramagnetic term becomes less negative or the carbon-6 more shielded. For the model to be consistent with this data it requires that fNHz < fOH < foMe < fF. Therefore, the modelisconsistent with the following order for mesomeric donor ability for the above substituents, NH, > OH > OMe > F. This order is in accord with previous concepts on the mesomeric donor ability of these substituents. Likewise, the same arguments hold for the X acceptor groups. When X is NOz, CHO, C02H, CN, or I, the Ivalues for these substituents can be considered reasonably constant. From eq 6 and ref 17, as f increases for a given I, upAAbecomes more shielded. For the model t o be consistent with the data f i o l < f C H 0 < fC02H < fcN < fI. That is, carbon-6 is more deshielded with respect to the halogen line in the following order: NO, > CHO > CO,H > CN > I. Therefore, the model implies that the order of mesomeric acceptor ability of the substituents is NOn > CHO > COzH > CN > I. Again, this order is the generally accepted order of the ability of these substituents t o act as mesomeric acceptor groups. In the preceding discussion, we have established a reasonable correlation between the mesomeric donor or acceptor ability of the substituent and deviations from the line drawn in Figure 3. Herein also lies a possible explanation for the difference between the
Ellis,et
ai.
1 lacNmr of 5-Substituted Uracils
4402
ortho carbon in substituted benzenes and carbon 6 in the corresponding uracils, that is, mesomeric delocalization. In substituted benzenes any charge that builds up on the ortho carbon can be delocalized further to the para carbon. However, in the substituted uracils the efficiency of the delocalization is considerably reduced with respect to benzene derivatives. This may be shown schematically in the following structures. The arrows in structures IV and V indicate possible pathways for charge delocalization, 0
0
N
V
for mesomeric donor and acceptor groups, respectively. The pathway indicated in IV would seem to be not very efficient, since it requires delocalization of electrons t o an already electron rich center. Therefore, C-6 in uracil derivatives (for donor X groups) would be more shielded than the ortho carbon in the substituted benzenes. Likewise, the pathway indicated by V may not be as efficient as the delocalization in the substituted benzenes, and hence carbon-6 would be less shielded (for X acceptor groups) than the ortho carbon in substituted benzenes. These trends with C-6 and C, are not unlike those that were observed by Maciell4 for vinyl carbons and C,. Therefore, these trends enforce our initial assumption that uracil derivatives can be considered as trisubstituted ethylenes. The remaining substituents that cannot be conveniently thought of as mesomeric donors or acceptors can be discussed in terms of their ability to polarize structures I1 and 111. For the substituents where X is given by H, CH3, and CHzOH, there is little effect on the chemical shift of (2-6. For Br and C1 there appears to be a cancellation of effects, that is, a small increase in I and a similar increment inf. This follows from the more detailed discussion of eq 5 and 6 given by Savitsky, et al. l7 C-5 in Substituted Uracils. The reliability of the predictions of the trends in the 13C chemical shifts for C-5 is somewhat lower than that of C-6, presumably because of the large changes in bonding that occur at C-5. These large changes in bonding, in all probability, will invalidate the assumption of a constant AE. In comparison, C-6 is relatively “isolated” from the substituent, whereas C-5 interacts with the substituent not only via the partial T bond formed between C-5 and X but also with the u bond to X. This situation does not allow an easy separation of the mesomeric effects, i.e., I constant and subsequent changes in f or vice versa. Furthermore, the neighbor anisotropy effects of the substituent are larger at C-5 than at C-6. These problems preclude a meaningfully detailed analysis of the trends exhibited in Figure 2. However, the qualitative discussion of the data in terms of possible fluctuations in I a n d f seems in order. It appears from Figure 2 and Table I that for the substituents F, OMe, OH, NOi, and NHz the C-5 becomes more shielded in the same order. Evidently, for these substituents, the inductive Q withdrawal of
these substituents dominates the observed trends as opposed to the mesomeric donor or acceptor ability of the substituent. For the substituents CH20H, CHj, C1, CHO, C02H, H, and Br there is an apparent cancellation of inductive effects and mesomeric effects. The increased shielding of C-5 for X equal to Br, CN, and I may be due to several factors, none of which are very obvious. It is common to attribute these shieldings to a neighbor anisotropy effect of these substituents. However, the range of chemical shifts observed in going from Br to I is approximately 27 ppm. This chemical shift difference is too large to be attributed to the neighbor anisotropy effect. It is well k n 0 w n ~ 8 ~ ~ ~ that the magnitude of this effect is independent of the nucleus involved. The range of proton chemical shifts is only approximately 10 ppm and, therefore, the magnitude of this neighboring group anisotropy effect cannot exceed this range. The effect of iodine as a substituent on l3C chemical shifts is just not understood. Correlation with Enzymatic Behavior. We sought to determine whether or not the 13C chemical shift data and our arguments for rationalizing this information could be extended to aid in understanding and evaluating the electronic component involved in the interaction of the uracils (or their corresponding nucleoside or nucleotide derivatives) with enzymatic systems. A convenient enzymatic case in point is that of thymidylate synthetase. 5-Fluor0-,~5-trifl~oromethyl-,~~ and 5-formyldeoxyuridine monophosphatez5 are potent inM ) while hibitors of thymidylate synthetase (Ki 5-hydroxymethyldeoxyuridine monophosphatez6 and thymidylate itself exhibit smaller inhibition constants (K,E 10-6 M ) which are characteristic of “so-called” product inhibitors. Finally, 5-HOdUMPz7 and 5 NHzdUMP**are strong inhibitors of cellular growth whose main site of action appears to be that of thymidylate synthetase. An analysis of the nmr data presented herein, especially the correlation depicted in Figure 3, reveals that the inhibitors of thymidylate synthetase fall into two categories represented by: (A) 5-flUOrO-, 5-hydroxy-, and 5-aminouracil, and (B) 5-formyl-, 5-trifluoromethyl-, 5-hydroxymethyl-, and 5-methyluracil (thymine). On close scrutiny, the C-5 substituents of the uracils in category A produce large, but similar chemical shift displacements when compared with uracil (C-4, 2.865.95; (2-5, -21.34 to -39.47; and C-6, 15.99-25.75) and feature C-5 heteroatom bonding systems. In comparison, the C-5 substituents of the uracils in category B are joined to C-5 by carbon-carbon linkages and elicit smaller chemical shift displacements from uracil (C-4, -0.32 to 4.56; C-5, - 1.58 to - 12.22; and C-6, -6.86 to 4.72). In other words, the I3C data indicate that the uracils in category A share certain similar properties with one another which are distinctly different from those of uracil and thymine,
=
(23) B. V. Cheney and D. M. Grant, J . Amer. Chem. Soc., 89, 5319 (1967). (24) P. Reyes and C. Heidelberger, Mol. Pharmacol., 1 , 1 4 (1965). (25) D. V. Santi and T. T. Sakai, Biochem. Biophys. Res. Commun., 42,813 (1971). (26) D. V. Santi and T. T. Sakai, ibid., 46,1320 (1972). (27) R . E. Beltz and D. W. Visser, J. Biol. Chem., 226, 1035 (1957). (28) M. Friedland and D. W. Visser. Biochim. Biophys. Acta, 51, 148 (1961).
Journal of the American Chemical Society 1 95:13 / June 27, 1973
4403
particularly with regard to the electronic conditions inferred from the 13C chemical shifts of C-5 and C-6. However, the uracils grouped in category B display 13C chemical shifts not unlike uracil itself. Thus, if extended t o the deoxynucleotide level, the analysis of the l3C data of the substituted uracils provides an electronic basis for differentiating among the inhibitors of thymidylate synthetase and is employed here to tentatively identify at least two mechanisms by which 5-substituted dUMP derivatives inhibit this enzyme. A current view of the initial steps in the reaction mechanism of thymidylate synthetase envisages the binding of dUMP to the active site of the enzyme followed by the attack of an enzyme-bound nucleophile, most probably a sulfhydryl group in the thiolate anion form, at the C-6 position of the substrate.29-31 If the behavior of the C-5 substituents of the uracils in category A is rationalized by considering -F, -NH2, and -OH as mesomeric donors, the latter substituents funnel electrons into the uracil ring system, causing a substantial increase in the n-electron charge density at C-6. Thus, we speculate that the inhibitory properties of the dUMP compounds derived from the uracils in category A result in part because the inhibitor satisfies the rather stringent structural requirements for substrate binding, but more importantly because the C-5 substituent is a mesomeric donor and causes an increase in n-electron density at C-6 which is not conducive to successful attack by the nucleophilic sulfhydryl group of the enzyme. The deoxynucleotide analogs of the uracils in category B include thymidylate and share structural and (29) D. V. Santi and C. F. Brewer, J . Amer. Chem. Soc., 90, 6236 (1968). (30) R. B. Dunlap, N. G . L. Harding, and F. M. Huennekens, Ann. N . Y.Acud. Sci., 186, 153 (1971). (31) T. I. Kalman, Biochemistry, 10,2567 (1971).
electronic properties with each other. However, 5FdUMP2j and 5-F3CdUMPZ4are 1000-fold better inhibitors of thymidylate synthetase than are thymidylate and 5-HOCH2dUMP. It is interesting to note that the C-6 chemical shifts of 5-formyluracil and 5 trifluoromethyluracil are both negative with respect to the C-6 of uracil itself. These data could be interpreted to indicate that C-6 of the corresponding irreversible inhibitors has a lower n-electron density than that of C-6 in dUMP; such a condition would make the C-6 of the inactivators more susceptible to nucleophilic attack than the corresponding carbon in dUMP. Thus, this 13C nmr investigation of 5-substituted uracils has suggested a means of categorizing deoxynucleotide inhibitors of thymidylate synthetase with regard to the electronic effects induced by their C-5 substituents. This technique is of potential value in predicting the effectiveness of substituted uracils as inhibitors of thymidylate synthetase before the more laborious procedure of testing on the deoxynucleotide level. We are presently extending such observations to include other enzymes which interact with pyrimidine bases, nucleosides, or nucleotides, and 13C nmr studies of the interaction of l 3C enriched substrates and inhibitors with these enzymes are now in progress. Acknowledgment. The XL-100-15 nmr spectrometer was purchased through funds made available to the Department of Chemistry uia a Department Development Grant by the National Science Foundation. P. D. E. is grateful to the University of South Carolina Committee on Research and Productive Scholarship for support of this research. This investigation was supported in part by Public Health Service Research Grant No. CA-12842 from the National Cancer Institute.
Ellis, et ai. 1
Nmr of5-Substituted Uracils
